Ouchterlony double immunodiffusion

Picture of an Ouchterlony double immunodiffusion plate, after immunodiffusion has taken place. In this, titre value of an antigen is quantified. The central well has an antibody, and the surrounding wells have decreasing concentration of the corresponding antigen.

Ouchterlony double immunodiffusion (also known as agar gel immunodiffusion or passive double immunodiffusion) is a simple, rather dated method which is still considered to be the gold standard for detection of extractable nuclear antigens. It is named after Örjan Ouchterlony, the Swedish physician who invented the test.

Procedure

A gel plate is cut to form a series of holes ("wells") in the gel. A sample extract of interest (for example human cells harvested from tonsil tissue) is placed in one well, and sera or purified antibodies are placed in another well and the plate left for 48 hours to develop. During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells. Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form what is known as an immune complex. This immune complex precipitates in the gel to give a thin white line, which is a visual signature of antigen recognition.

The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple wells with different antibodies or mixtures of antibodies, and antigen-antibody reactivity can be seen by observing between which wells the precipitate is observed. When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected. The zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely).

Theory

Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of Immunoglobulin M there is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed. Experimentally, an increasing amount of antigen is added to a constant amount of antibody in solution. Initially at low antigen concentration, all of the antigen is contained in the precipitate. This is called the antibody-excess zone (i.e. prozone phenomenon). As more antigen is added, the amount of protein precipitated increases until the antigen/antibody molecules are at an optimal ratio. This is known as the zone of equivalence or equivalence point. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease. This is known as the antigen excess zone.

External links

• Simulator at University of California, Irvine
• Overview at University of Maryland